EP1007648A1

EP1007648A1 - Anti-obesity proteins

Info

Publication number: EP1007648A1
Application number: EP97954183A
Authority: EP
Inventors: John M. Beals; James A. Hoffmann; Allen D. Kline; Richard W. Schevitz; Faming Zhang
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 1996-12-20
Filing date: 1997-12-19
Publication date: 2000-06-14
Also published as: WO1998028414A1; CA2274799A1; AU5802898A; EP1007648A4; JP2001507228A

Abstract

The present invention provides anti-obesity proteins, which when administered to a patient regulate fat tissue. Accordingly, such agents allow patients to overcome their obesity handicap and live normal lives with much reduced risk for type 2 diabetes, cardiovascular disease, and cancer.

Description

ANTI-OBESITY PROTEINS

Field of the Invention

The present invention is in the field of human medicine, particularly in the treatment of obesity and disorders associated with obesity. Most specifically the invention relates to anti-obesity proteins that when administered to a patient regulate fat tissue.

Background of the Invention

Obesity, and especially upper body obesity, is a common and very serious public health problem in the United States and throughout the world. According to recent statistics, more than 25% of the United States population and 27% of the Canadian population are overweight

[Kuczmarski, R. J. , Amer. J. of Clin . Nutr. 55:495S-502S (1992); Reeder, B. A., et. al . , Can . Med. Assoc . J. , 146:2009-2019 (1992)]. Upper body obesity is the strongest risk factor known for people with type 2 diabetes, and is a strong risk factor for cardiovascular disease and cancer as well. Recent estimates for the medical cost of obesity are $150,000,000,000 world wide. The problem has become serious enough that the surgeon general has begun an initiative to combat the ever increasing adiposity rampant in American society.

Much of this obesity-induced pathology can be attributed to the strong association with dyslipidemia, hypertension, and insulin resistance. Many studies have demonstrated that reduction in obesity by diet and exercise reduces these risk factors dramatically. Unfortunately, these treatments are largely unsuccessful, with a failure rate reaching 95%. This failure may be due to the fact that the condition is strongly associated with genetically inherited factors that contribute to increased appetite, preference for highly caloric foods, reduced physical activity, and increased lipogenic metabolism. This indicates that people inheriting these genetic traits are prone to becoming obese regardless of their efforts to combat the condition. Therefore, a pharmacological agent that can correct this adiposity handicap and allow the physician to successfully treat obese patients in spite of their genetic inheritance is needed.

Physiologists have postulated for years that, when a mammal overeats, the resulting excess fat signals to the brain that the body is obese, which in turn causes the body to eat less and burn more fuel [Hervey, G. R., Nature 222:629-631 (1969)]. This "feedback" model is supported by parabiotic experiments, which implicate a circulating hormone controlling adiposity.

The ob/ob mouse is a model of obesity and diabetes that is known to carry an autosomal recessive trait linked to a mutation in the sixth chromosome. Recently, Zhang, Y. and co-workers published the positional cloning of the mouse gene linked with this condition [Zhang, Y., et al . Nature 372:425-432 (1994)]. This report disclosed a gene coding for a 167 amino acid protein with a 21 amino acid signal peptide that is exclusively expressed in adipose tissue. The rat obese gene was subsequently cloned and expressed [Murakami, T. et al . , Biochem . Biophys . Res . Comm . 209:944- 952 (1995)] . The protein, which is apparently encoded by the ob gene, is now speculated to be the adiposity regulating hormone.

The protein encoded by the ob gene is pharmacologically active, but the physical properties of the protein are less than desirable. The human protein, for example, is prone to precipitation and aggregation in both a formulation and under physiological conditions.

Formulations of a protein containing a precipitate, or that permit precipitation at the site of administration, increase the risk of producing an immunological response in the patient and may result in irritation at the site of injection. Additives to the formulation, such as detergents or surfactants, are undesirable because of increased risk of toxicology. Accordingly, there remains a need to develop pharmacological agents that demonstrate pharmacological activity and provide improved physical and chemical stability. Applicants have discovered that the aggregation observed in the native human protein is due, in part, to hydrophobic interactions at the surface of the protein, particularly at residues 1, 2, 3, 30, 36, 41, 42, 43, 45, 46, 47, 48, 49, 50, 74, 89, 92, 99, 100, 138, and 142. Applicants have further discovered that, by substituting these positions or regions of the protein with charged or hydrophilic amino acids, the propensity of the ob protein to aggregate is dramatically reduced. This reduction in aggregation provides a much improved pharmacological agent. Accordingly, the present invention provides biologically active obesity proteins. Such agents allow patients to overcome their obesity handicap and live normal lives with a more normalized risk for type 2 diabetes, cardiovascular disease, and cancer.

Summary of Invention The present invention is directed to a protein of the Formula (I) :

5 10 15 Xaa Xaa lie Gin Lys Val Gin Asp Asp Thr Lys Thr Leu lie Lys Thr

20 25 30 lie Val Thr Arg lie Xaa Asp lie Ser His Thr Xaa Ser Val Ser Ser 35 40 45

Lys Gin Lys Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro lie

50 55 60

Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin lie

65 70 75 80

Leu Thr Ser Met Pro Ser Arg Xaa Xaa lie Gin lie Ser Asn Asp Leu

85 90 95 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys

100 105 110

His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser . Thr Glu Val Val Ala Leu Ser Arg

130 135 140

Leu Gin Gly Ser Leu Gin Asp Met Leu Xaa Gin Leu Asp Leu Ser Pro

145

Gly Cys ( SEQ ID NO : l ) ( I ) wherein:

Xaa at position 1 is Val or absent; Xaa at position 2 is Pro or absent;

Xaa at position 22 is Asn or Ser;

Xaa at position 28 is Gin or absent;

Xaa at position 72 is Asn, Gin, Glu or Asp;

Xaa at position 73 is Val or Met; Xaa at position 100 is Trp, Gin, Glu, Asp, Ser,

Thr, Lys, His, or Arg;

Xaa at position 138 is Trp, Gin, Glu, Asp, Ser, Thr, Lys, His, or Arg; said protein having at least one of the following substitutions:

Xaa at position 1 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg;

Xaa at position 2 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg; lie at position 3 is replaced with Glu, Asp, Arg,

Lys, or His;

Val at position 30 is replaced with Glu, Asp, Arg, Lys, or His;

Val at position 36 is replaced with Glu, Asp, Arg, Lys, or His;

Phe at position 41 is replaced with Glu, Asp, Arg, Lys, or His; lie at position 42 is replaced with Glu, Asp, Arg, Lys, or His; Pro at position 43 is replaced with Glu, Asp, Arg,

Lys, or His;

Leu at position 45 is replaced with Glu, Asp, Arg, Lys, or His; His at position 46 is replaced with Glu, Asp, Arg, or Lys;

Pro at position 47 is replaced with Glu, Asp, Arg, Lys, or His; lie at position 48 is replaced with Glu, Asp, Arg,

Lys, or His;

Leu at position 49 is replaced with Glu, Asp, Arg, Lys, or His;

Thr at position 50 is replaced with Glu, Asp, Arg, Lys, or His; lie at position 74 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser;

Val at position 89 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; Phe at position 92 is replaced with Gin, Glu, Asp,

Arg, Lys, His, Thr or Ser_f- Pro at position 99 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; or

Leu at position 142 is replaced with Glu, Asp, Arg, Lys, or His; or a pharmaceutically acceptable salt thereof .

The invention further provides a method of treating obesity or those conditions associated with obesity, which comprises administering to a mammal in need thereof a protein of the Formula (I) .

The invention further provides a pharmaceutical formulation, which comprises a protein of the Formula (I) , optionally having a leader sequence, together with one or more pharmaceutically acceptable diluents, carriers, or excipients therefor.

The invention further provides proteins of the Formula (I) having additionally a leader sequence bonded to the N-terminus of the protein of Formula I. Such proteins are useful for their anti-obesity activity and as intermediates in the preparation of proteins of the Formula (I) . The invention further provides DNA encoding a protein of Formula (I) , or a protein of Formula (I) having a leader sequence.

An additional embodiment of the present invention is a process for producing a protein of Formula (I) , which comprises :

(a) transforming a host cell with DNA that encodes the protein of Formula (I) or a protein of Formula (I) having a leader sequence; (b) culturing the host cell under conditions permitting expression of the protein;

(c) recovering the expressed protein; and, optionally,

(d) cleaving enzymatically the leader sequence. The invention further provides a protein for use in treating obesity and conditions associated with obesity, as well as, for use in preparing a medicament used to treat obesity and conditions associated with obesity.

Detailed Description

For purposes of the present invention, as disclosed and claimed herein, the following terms and abbreviations are defined as follows:

Base pair (bp) -- refers to DNA or RNA. The abbreviations A,C,G, and T correspond to the 5'- monophosphate forms of the nucleotides (deoxy) adenine, (deoxy) cytidine, (deoxy) guanine, and (deoxy) thymine, respectively, when they occur in DNA molecules. The abbreviations U,C,G, and T correspond to the 5¹- monophosphate forms of the nucleosides uracil, cytidine, guanine, and thymine, respectively, when they occur in RNA molecules. In double stranded DNA, base pair may refer to a partnership of A with T or C with G. In a DNA/RNA heteroduplex, base pair may refer to a partnership of T or U with A or C with G. FMOC - an abbreviation for 9- fluorenymethoxycarbony.

Immunoreactive Protein (s) -- a term used to collectively describe antibodies, fragments of antibodies capable of binding antigens of a similar nature as the parent antibody molecule from which they are derived, and single chain polypeptide binding molecules [Bird, E. R., et al . , PCT Application No. PCT/US 87/02208, International Publication No. WO 88/01649, published March 10, 1988] . Plasmid -- an extrachromosomal self-replicating genetic element .

PAM - an abbreviation for 4-hydroxymethyl- phenylacetamidomethyl .

PMSF -- an abbreviation for phenylmethylsulfonyl fluoride.

Reading frame -- the nucleotide sequence from which translation occurs "read" in triplets by the translational apparatus of tRNA, ribosomes and associated factors, each triplet corresponding to a particular amino acid. Because each triplet is distinct and of the same length, the coding sequence must be a multiple of three. A base pair insertion or deletion (termed a frameshift mutation) may result in two different proteins being coded for by the same DNA segment. To insure against this, the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, i.e., the correct "reading frame" must be maintained.

Recombinant DNA Cloning Vector -- any autonomously replicating agent including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.

Recombinant DNA Expression Vector -- any recombinant DNA cloning vector in which a promoter has been incorporated. Replicon -- A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector.

TFA - an abbreviation for trifluoroacetic acid. Transcription -- the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence.

Translation -- the process whereby the genetic information of messenger RNA is used to specify and direct the synthesis of a polypeptide chain.

* Tris -- an abbreviation for tris (hydroxymethyl) - aminomethane .

Treating -- describes the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of a protein of present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. Treating obesity, therefore, includes the inhibition of food intake, the inhibition of weight gain, and inducing weight loss in patients in need thereof.

Vector -- a replicon used for the transformation of cells in gene manipulation bearing polynucleotide sequences corresponding to appropriate protein molecules which, when combined with appropriate control sequences, confer specific properties on the host cell to be transformed. Plasmids, viruses, and bacteriophage are suitable vectors, since they are replicons in their own right. Artificial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases. Vectors include Recombinant DNA cloning vectors and Recombinant DNA expression vectors .

X-gal -- an abbreviation for 5-bromo-4-chloro-3- indolyl beta-D-galactoside. The amino acid abbreviations are accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. § 1.822 (b)(2) (1993). Unless otherwise indicated the amino acids are in the L configuration.

As noted above the present invention provides a protein of the Formula (I) :

5 10 15

Xaa Xaa lie Gin Lys Val Gin Asp Asp Thr Lys Thr Leu lie Lys Thr

20 25 30

He Val Thr Arg He Xaa Asp He Ser His Thr Xaa Ser Val Ser Ser

35 40 45 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He

50 55 60

Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 65 70 75 80

Leu Thr Ser Met Pro Ser Arg Xaa Xaa He Gin He Ser Asn Asp Leu

85 90 95

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys

100 105 110

His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly

115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg

130 135 140

Leu Gin Gly Ser Leu Gin Asp Met Leu Xaa Gin Leu Asp Leu Ser Pro 145

Gly Cys (SEQ ID NO:l) (I) wherein:

Xaa at position 1 is Val or absent;

Xaa at position 2 is Pro or absent; Xaa at position 22 is Asn or Ser;

Xaa at position 28 is Gin or absent;

Xaa at position 72 is Asn, Gin, Glu or Asp;

Xaa at position 73 is Val or Met;

Xaa at position 100 is Trp, Gin, Glu, Asp, Ser, Thr, Lys, His, or Arg;

Xaa at position 138 is Trp, Gin, Glu, Asp, Ser,

Thr, Lys, His, or Arg; said protein having at least one of the following substitutions : Xaa at position 1 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg;

Lys, or His;

Val at position 30 is replaced with Glu, Asp, Arg, Lys, or His;

Val at position 36 is replaced with Glu, Asp, Arg, Lys, or His;

Lys, or His;

Leu at position 45 is replaced with Glu, Asp, Arg, Lys, or His;

His at position 46 is replaced with Glu, Asp, Arg, or Lys;

Pro at position 47 is replaced with Glu, Asp, Arg, Lys, or His; lie at position 48 is replaced with Glu, Asp, Arg, Lys, or His; Leu at position 49 is replaced with Glu, Asp, Arg,

Lys, or His;

Val at position 89 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser;

Phe at position 92 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; Pro at position 99 is replaced with Gin, Glu, Asp,

Arg, Lys, His, Thr or Ser; or Leu at position 142 is replaced with Glu, Asp, Arg, Lys, or His; or a pharmaceutically acceptable salt thereof.

Preferred proteins are those having one to three substitutions; most preferably one or two substitutions defined in Formula (I) .

Other preferred proteins of the present invention are those of SEQ ID N0:1, wherein:

Val at position 36 is replaced with Arg, Lys, or His; lie at position 74 is replaced with Glu or Asp, or Phe at position 92 is replaced with Arg, Lys, or

His.

Other preferred proteins of the present invention are those of SEQ ID NO:l, wherein:

Phe at position 92 is replaced with Asp, Glu, Lys, Arg, or His, or pharmaceutically acceptable salts thereof.

Other preferred proteins of the present invention are those of SEQ ID NO:l, wherein: Phe at position 92 is replaced with Asp or Glu, or pharmaceutically acceptable salts thereof.

Xaa at position 1 is Val or absent; Xaa at position 2 is Pro or absent;

Xaa at position 22 is Asn; Xaa at position 28 is Gin or absent; Xaa at position 72 is Asn; Xaa at position 73 is Val; Xaa at position 100 is Trp, Glu, Asp, Ser, Thr,

Lys, His, or Arg;

Xaa at position 138 is Trp, Glu, Asp, Ser, Thr, Lys, His, or Arg; said protein having at least one of the following substitutions: Leu at position 45 is replaced with Glu, Asp, Arg, Lys, or His;

His at position 46 is replaced with Glu, Asp, Arg, or Lys; Pro at position 47 is replaced with Glu, Asp, Arg,

Lys, or His; lie at position 48 is replaced with Glu, Asp, Arg, Lys, or His; or

Leu at position 142 is replaced with Glu, Asp, Arg, Lys, or His; or pharmaceutically acceptable salts thereof.

Xaa at position 1 is Val; Xaa at position 2 is Pro;

Xaa at position 28 is Gin or absent;

Xaa at position 72 is Asn;

Xaa at position 100 is Trp, Glu, or Asp;

Xaa at position 138 is Trp, Glu or Asp; said protein having at least one of the following substitutions :

His at position 46 is replaced with Glu, Asp, Arg, or Lys; lie at position 48 is replaced with Glu, Asp, Arg, Lys, or His; or

Phe at position 92 is replaced with Glu, Asp, Arg, Lys, or His; or pharmaceutically acceptable salts thereof .

Xaa at position 1 is Val; Xaa at position 2 is Pro; Xaa at position 28 is Gin or absent; Xaa at position 72 is Asn; Xaa at position 100 is Trp, Glu, or Asp;

His at position 46 is replaced with Glu, Asp, Arg, or Lys; or He at position 48 is replaced with Glu, Asp, Arg,

Lys, or His; or pharmaceutically acceptable salts thereof.

Still more preferred are proteins of the present invention are those of SEQ ID N0:1, wherein: Xaa at position 1 is Val;

Xaa at position 2 is Pro;

Xaa at position 28 is Gin or absent;

Xaa at position 72 is Asn;

Xaa at position 100 is Trp or Asp; Xaa at position 138 is Trp; said protein having at least one of the following substitutions :

His at position 46 is replaced with Glu, Asp, Arg, or Lys; He at position 48 is replaced with Asp, Arg, Lys, or His; or

Phe at position 92 is replaced with Asp, Glu, Arg, Lys, or His; or pharmaceutically acceptable salts thereof . Still more preferred are proteins of the present invention are those of SEQ ID NO:l, wherein:

Xaa at position 1 is Val;

Xaa at position 2 is Pro;

Xaa at position 28 is Gin or absent; Xaa at position 72 is Asn;

Xaa at position 100 is Trp or Asp;

Xaa at position 138 is Trp; said protein having at least one of the following substitutions : His at position 46 is replaced with Glu, Asp, Arg, or Lys ; or He at position 48 is replaced with Asp, Arg, Lys, or His; or pharmaceutically acceptable salts thereof .

Xaa at position 1 is Val or absent;

Xaa at position 2 is Pro or absent;

Xaa at position 28 is Gin or absent;

Xaa at position 72 is Asn, Glu or Asp; Xaa at position 100 is Trp, Glu, Asp, Ser, Thr,

Lys, His, or Arg;

Xaa at position 138 is Trp, Glu, Asp, Ser, Thr, Lys, His, or Arg; said protein having at least one of the following substitutions:

Xaa at position 1 is replaced with Glu, Asp, Ser, Thr, Lys, His, Arg, or absent;

Xaa at position 2 is replaced with Glu, Asp, Ser, Thr, Lys, His, Arg, or absent; Val at position 89 is replaced with Glu, Asp, Arg,

Lys, or His; or

Phe at position 92 is replaced with Glu, Asp, Arg, Lys, or His; or a pharmaceutically acceptable salt thereof . Other preferred proteins are those of Formula I wherein:

Xaa at position 1 is Val or absent;

Xaa at position 2 is Pro or absent;

Xaa at position 28 is Gin or absent; Xaa at position 72 is Asn, Glu or Asp;

Xaa at position 100 is Glu, Asp, Lys, His, or Arg;

Xaa at position 138 is Trp, Glu, Asp, Ser, Thr, Lys, His, or Arg; said protein having at least one of the following substitutions: Phe at position 92 is replaced with Glu, Asp , Arg, Lys , or His ;

Pro at position 99 is replaced with Glu, Asp , Arg, Lys , or His ; or a pharmaceutically acceptable salt thereof .

Preferred species within Formula I include species of SEQ ID NO : 2 through SEQ ID NO : 7 :

5 10 15

Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr

20 25 30

He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser

35 40 45 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu Asp Pro He

50 55 60

Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He

65 70 75 80

Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu

85 90 95

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys

100 105 110

His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly

115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg

130 135 140

Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro

145 Gly Cys (SEQ ID NO: 2i

5 10 15

Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr

20 25 30

He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser

35 40 45 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro Asp

50 55 60

Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 65 70 75 80

Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu

85 90 95

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys

100 105 110

His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125

Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg

130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro

145

Gly Cys (SEQ ID NO: 3)

5 10 15

Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr

20 25 30

He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser

35 40 45

Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu Asp Pro He

50 55 60 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He

65 70 75 80

Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 85 90 95

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys

100 105 110

His Leu Pro Asp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly

115 120 125

Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg

130 135 140 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro

145

Gly Cys (SEQ ID NO: 4)

5 10 15

Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr

20 25 30

He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser

35 40 45

Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro Asp

50 55 60 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 65 70 75 80

Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu

85 90 95 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys

100 105 110

His Leu Pro Asp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125

Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg

130 135 140

Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro

145 Gly Cys (SEQ ID NO: 5)

5 10 15

Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr

20 25 30

He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser

35 40 45 Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He

50 55 60

Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 65 70 75 80

Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu

85 90 95

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Asp Ser Lys Ser Cys

100 105 110

His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly

115 120 125 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg

130 135 140

Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145

Gly Cys (SEQ ID NO: 6)

5 10 15

Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 20 25 30

He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser

35 40 45

Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 50 55 60

Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He

65 70 75 80 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu

85 90 95

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Asp Ser Lys Ser Cys 100 105 110

His Leu Pro Asp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly

115 120 125

Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg

130 135 140

Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro

145 Gly Cys (SEQ ID NO: 7)

As mentioned above, the invention also provides proteins of the Formula ( I) having additionally a leader sequence bonded to the N- terminus of the protein of Formula I . Such proteins are useful for their anti-obesity activity and as intermediates in the preparation of proteins of the Formula (I) . The species having SEQ ID NO : 8-13 are particularly preferred proteins of Formula (I) having the leader sequence Met -Arg bonded to the N- terminus of the proteins of SEQ ID NO : 2 -7 , respectively.

5 10 15

Met Arg Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He

20 25 30 Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val

35 40 45

Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu Asp 50 55 60

Pro He Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin

65 70 75 80

Gin He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn

85 90 95

Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys

100 105 110 Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu

115 120 125

Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu 130 135 140

Ser Arg Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu

145

Ser Pro Gly Cys (SEQ ID NO: 8)

5 10 15

Met Arg Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He 20 25 30

Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val

35 40 45

Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His

50 55 60

Pro Asp Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin

65 70 75 80 Gin He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn

85 90 95

Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys 100 105 110

Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu

115 120 125

Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu

130 135 140

Ser Arg Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu

145 Ser Pro Gly Cys (SEQ ID NO: 9)

5 10 15

Met Arg Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He

20 25 30 Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val

35 40 45

Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu Asp 50 55 60

Pro He Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin

65 70 75 80

Gin He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn

85 90 95

Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys 100 .105 110

Ser Cys His Leu Pro Asp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu

115 120 125

Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu

130 135 140

Ser Arg Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu 145

Ser Pro Gly Cys (SEQ ID NO: 10)

5 10 15

Met Arg Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He

20 25 30

Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val

35 40 45 Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His

50 55 60

Pro Asp Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin 65 70 75 80

Gin He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn

85 90 95

Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys

100 105 110

Ser Cys His Leu Pro Asp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu

115 120 125 Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu

130 135 140

Ser Arg Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu 145

Ser Pro Gly Cys (SEQ ID NO: 11)

5 10 15

Met Arg Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He

20 25 30

Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val

35 40 45 Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His

50 55 60

Pro He Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin 65 70 75 80

Gin He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn 85 90 95

Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Asp Ser Lys

100 105 110 Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu

115 120 125

Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu 130 135 140

Ser Arg Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu

145

Ser Pro Gly Cys (SEQ ID NO: 12)

5 10 15

Met Arg Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He

20 25 30 Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val

35 40 45

Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His 50 55 60

Pro He Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin

65 70 75 80

Gin He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn

85 90 95

Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Asp Ser Lys

100 105 110 Ser Cys His Leu Pro Asp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu

115 120 125

Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu 130 135 140

Ser Arg Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu

145

Ser Pro Gly Cys (SEQ ID NO: 13)

The present invention provides biologically active proteins that provide effective treatment for obesity. Applicants have discovered that specific substitutions to the hydrophobic residues on the surface of the protein result in improved properties. These residues could not be predicted from the primary sequence . The proteins having these substitutions are pharmacologically active and have a reduced propensity to aggregate when compared to both the mouse and human forms of the protein. The present invention permits obesity protein to be more readily formulated at higher concentrations, and increases their pharmaceutical elegance, because they are compatible with commonly-used preservatives.

The claimed proteins ordinarily are prepared by recombinant techniques . Techniques for making substitutional mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis. The mutations that might be made in the DNA encoding the present anti-obesity proteins must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure [DeBoer, H.A., et al . , European Patent Publication No. 75,444 A2 , published March 30, 1983] . The proteins of the present invention may be produced either by recombinant DNA technology or well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods .

A. Solid Phase

The synthesis of the claimed proteins may proceed by solid phase peptide synthesis or by recombinant methods. The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area such as Dugas, H. and Penney, C, Bioorganic Chemistry Springer-Verlag, New York, (1981) 54- 59. For example, peptides may be synthesized by solid-phase methodology utilizing an PE-Applied Biosystems 433A peptide synthesizer (Perkin Elmer-Applied Biosystems Division, Foster City, California) and synthesis cycles supplied by Applied Biosystems. Boc amino acids and other reagents are commercially available from PE-Applied Biosystems and other chemical supply houses. Sequential Boc chemistry using double couple protocols are applied to the starting p-methyl benzhydryl amine resins for the production of C-terminal carboxamides . For the production of C-terminal acids, the corresponding PAM resin is used. Arginine, Asparagine, Glutamine, Histidine, and Methionine are coupled using preformed hydroxy benzotriazole esters. The following side chain protection may be used:

Arg, Tosyl

Asp, cyclohexyl or benzyl Cys, 4-methylbenzyl

Glu, cyclohexyl

His, benzyloxymethyl

Lys, 2-chlorobenzyloxycarbonyl

Met, sulfoxide Ser, Benzyl

Thr, Benzyl

Trp, formyl

Tyr, 4-bromo carbobenzoxy

Boc deprotection may be accomplished with trifluoroacetic acid (TFA) in methylene chloride. Formyl removal from Trp is accomplished by treatment of the peptidyl resin with 20% piperidine in dimethylformamide for 60 minutes at 4°C. Met (0) can be reduced by treatment of the peptidyl resin with TFA/dimethylsulfide/conc. HC1 (95/5/1) at 25°C for 60 minutes. Following the above pre-treatments, the peptides may be further deprotected and cleaved from the resin with anhydrous hydrogen fluoride containing a mixture of 10% m- cresol or m-cresol/10% p-thiocresol or m-cresol/p- thiocresol/dimethyl-sulfide. Cleavage of the side chain protecting group (s) and of the peptide from the resin is carried out at 0 C or below, preferably -20°C for thirty minutes followed by thirty minutes at 0°C. After removal of the HF, the peptide/resin is washed with ether. The peptide is extracted with glacial acetic acid and lyophilized. Purification is accomplished by reversed-phase C18 chromatography in 0.1% TFA with a gradient of increasing acetonitrile concentration, e . g. , a 2.2 cm X 25 cm Vydac™ column (The Separations Group, Inc., Hesperia, CA) .

One skilled in the art recognizes that the solid phase synthesis could also be accomplished using the FMOC strategy and a TFA/scavenger cleavage mixture.

B. Recombinant Synthesis

The claimed proteins may also be produced by recombinant methods. Recombinant methods are preferred if a high yield or if specific post-translational modifications are desired. The basic steps in the recombinant production of protein include : a) construction of a synthetic or semi-synthetic (or isolation from natural sources) DNA encoding the claimed protein, b) integrating the coding sequence into an expression vector in a manner suitable for the expression of the protein either alone or as a fusion protein, c) transforming an appropriate eukaryotic or prokaryotic host cell with the expression vector, and d) recovering and purifying the recombinantly produced protein. a. Gene Construction

Synthetic genes, the in vitro or in vivo transcription and translation of which will result in the production of the protein may be constructed by techniques well known in the art . Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences may be constructed which encode the claimed proteins . In the preferred practice of the invention, synthesis is achieved by recombinant DNA technology. Methodology of synthetic gene construction is well known in the art [Brown, E. L., et al . Methods in Enzymology, Academic Press, New York, NY, 68:109-151 (1979)] . The DNA sequence corresponding to the synthetic claimed protein gene may be generated using conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A or 380B DNA synthesizers (Perkin Elmer, Applied Biosystems Division, Foster City, California) .

It may desirable in some applications to modify the coding sequence of the claimed protein so as to incorporate a convenient protease sensitive cleavage site, e.g., between the signal peptide and the structural protein facilitating the controlled excision of the signal peptide from the fusion protein construct.

The gene encoding the claimed protein may also be created by using polymerase chain reaction (PCR) . The template can be a cDNA library (commercially available from CLONETECH or STRATAGENE) or itiRNA isolated from human adipose tissue. Such methodologies are well known in the art. See, e . g. , Maniatis, T., et al . Molecular Cloning: A Laboratory Manual , 2^nd Ed., Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989) . b. Direct Expression or Fusion Protein The claimed protein may be made either by direct expression or as fusion protein comprising the claimed protein followed by enzymatic or chemical cleavage. A variety of peptidases (e.g., trypsin) which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g., diaminopeptidase) of the peptide chain are known. Furthermore, particular chemicals (e.g., cyanogen bromide) will cleave a polypeptide chain at specific sites. The skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See, e . g. , Carter, P., Chapter 13 in Protein Purification : From Molecular Mechanisms to Large- Scale Processes, Ladisch, M., et al . (Eds.) American Chemical Soc, Washington, D.C. (1990). c. Vector Construction

Construction of suitable vectors containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to form the plasmids required.

To effect the translation of the desired protein, one inserts the engineered synthetic DNA sequence in any of a plethora of appropriate recombinant DNA expression vectors through the use of appropriate restriction endonucleases . A synthetic coding sequence is designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into these expression and amplification and expression plasmids. The isolated cDNA coding sequence may be readily modified by the use of synthetic linkers to facilitate the incorporation of this sequence into the desired cloning vectors by techniques well known in the art. The particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed. Restriction sites are chosen so as to properly orient the coding sequence with control sequences to achieve proper in-frame reading and expression of the claimed protein.

In general, plasmid vectors containing promoters and control sequences which are derived from species compatible with the host cell are used with these hosts. The vector ordinarily carries a replication site as well as marker sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species [Bolivar, F., et al . , Gene 2:95-113 (1977)] . Plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DNA technology. The desired coding sequence is inserted into an expression vector in the proper orientation to be transcribed from a promoter and ribosome binding site, both of which should be functional in the host cell in which the protein is to be expressed. An example of such an expression vector is a plasmid described in Belagaje, R. M., et al . , U.S. patent No. 5,304,473, issued, April 19, 1994, the teachings of which are herein incorporated by reference. The gene encoding A-C-B proinsulin described in U.S. Patent No. 5,304,473 can be removed from the plasmid pRB182 with restriction enzymes Ndel and BamHI. The genes encoding the protein of the present invention can be inserted into the plasmid backbone on a Ndel/BamHI restriction fragment cassette . d. Prokaryotic Expression In general, prokaryotes are used for cloning of

DNA sequences in constructing the vectors useful in the invention. For example, E. coli K12 strain 294 (ATCC No. 31446) is particularly useful. Other microbial strains which may be used include E. coli B and E. coli X1776 (ATCC No. 31537) . These examples are illustrative rather than limiting.

Prokaryotes also are used for expression. The aforementioned strains, as well as E. coli W3110 (prototrophic, ATCC No. 27325) , bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans, and various pseudomonas species may be used. Promoters suitable for use with prokaryotic hosts include the β-lactamase (vector pGX2907 [ATCC 39344] contains the replicon and β-lactamase gene) and lactose promoter systems [Chang, A. C. Y., et al . , Nature, 275:617-624 (1978); and Goeddel, D. V., et al., Nature 281:544-548 (1979)], alkaline phosphatase, the tryptophan (trp) promoter system (vector pATHl [ATCC 37695] is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter) and hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC-37282) . However, other functional bacterial promoters, whose nucleotide sequences are generally known, enable one skilled in the art to ligate them to DNA encoding the protein using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding protein. e. Eukaryotic Expression

The protein may be recombinantly produced in eukaryotic expression systems. Preferred promoters controlling transcription in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40) , adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g., β-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication [Fiers, W., et al., Nature, 273:113- 120 (1978)] . The entire SV40 genome may be obtained from plasmid pBRSV, ATCC 45019. The immediate early promoter of the human cytomegalovirus may be obtained from plasmid pCMBβ (ATCC 77177) . Of course, promoters from the host cell or related species also are useful herein. Transcription of a DΝA encoding the claimed protein by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DΝA, usually about 10-300 bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent having been found 5' [Laimins, L. A., et al . , Proc . Nat ' l Acad . Sci . (USA) 78:464-468 (1981)] and 3' [Lusky, M. , et al . , Mol . Cell . Bio . 3:1108-1122 (1983)] to the transcription unit, within an intron [Banerji, J., et al., Cell 23 : 129-140 (1983)] as well as within the coding sequence itself [Osborne, T. F., et al . , Mol . Cell . Bio. 4:1293-1305 (1984)] . Many enhancer sequences are now known from mammalian genes (globin, RSV, SV40, EMC, elastase, albumin, α-fetoprotein, and insulin) . Typically, however, one will use an enhancer from a eukaryotic cell virus . Examples include the SV40 late enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding protein. The 3¹ untranslated regions also include transcription termination sites.

Expression vectors may contain a selection gene, also termed a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR, which may be derived from the Bglll/Hindlll restriction fragment of pJOD-10 [ATCC 68815] ) , thymidine kinase (herpes simplex virus thymidine kinase is contained on the BamHI fragment of vP-5 clone (ATCC 2028) , or neomycin (G418) resistance genes, which are obtainable from pNN414 yeast artificial chromosome vector (ATCC 37682) . When such selectable markers are successfully transferred into a mammalian host cell, the transfected mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow without a supplemented media. Two examples are: CHO DHFR" cells (ATCC CRL-9096) and mouse LTK" cells [L-M(TK-) ATCC CCL-2.3] . These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine . Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell . Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, [Southern P J., et al., J^". Molec. Appl . Genet . 1:327-341

(1982)], mycophenolic acid [Mulligan, R. C. et al . , Science 209:1422-1427 (1980)], or hygromycin [Sugden, B. et al . , Mol Cell . Biol . 5:410-413 (1985)]. The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug, such as, G418, neomycin (geneticin) , xgpt (mycophenolic acid) , or hygromycin, respectively.

A preferred vector for eukaryotic expression is pRc/CMV. pRc/CMV is commercially available from Invitrogen Corporation, San Diego, CA. To confirm correct sequences in constructed plasmids, the ligation mixtures are used to transform E. coli K12 strain DH5a (ATCC 31446) and successful transformants are selected by antibiotic resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction and/or sequenced by the method of. Messing, J., et al . , Nucleic Acids Res . 9:309-321 (1981).

Host cells may be transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. The techniques of transforming cells with the aforementioned vectors are well known in the art and may be found in such general references as Maniatis, T., et al. Molecular Cloning: A Laboratory Manual , 2^nd Ed., Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989) , or Current Protocols in Molecular Biology (1989) and supplements.

Preferred suitable host cells for expressing the vectors encoding the claimed proteins in higher eukaryotes include: African green monkey kidney cell line transformed by SV40 (COS-7, ATCC CRL-1651) ; transformed human primary embryonal kidney cell line 293 [Graham, F. L. et al . , J. Gen Virol . 36:59-72 (1977); Harrison, T., et al . , Virology 77:319-329 (1977); Graham, F. L. et al . , Virology 86:10-21 (1978)]; baby hamster kidney cells [BHK-21 (C-13) , ATCC CCL- 10; MacPherson, I., et al , Virology 16 ;147-151 (1962)]; Chinese hamster ovary cells [CHO-DHFR- (ATCC CRL-9096)]; mouse Sertoli cells [TM4, ATCC CRL-1715; Mather, J. P., Biol . Reprod. 23:243-252 (1980)]; African green monkey kidney cells (VERO 76, ATCC CRL-1587) ; human cervical epitheloid carcinoma cells (HeLa, ATCC CCL-2) ; canine kidney cells (MDCK, ATCC CCL-34) ; buffalo rat liver cells (BRL 3A, ATCC CRL-1442) ; human diploid lung cells (WI-38, ATCC CCL- 75); human hepatocellular carcinoma cells (Hep G2, ATCC HB- 8065) ; and mouse mammary tumor cells (MMT 060562, ATCC CCL51) . f . Yeast Expression In addition to prokaryotic and mammalian host cells, eukaryotic microorganisms such as yeast may also be used as host cells. Saccharomyces cerevisiae, common baker's yeast, is the most commonly-used eukaryotic microorganism for expressing heterologous proteins, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used [ATCC-40053, Stinchcomb, D. T., et al . , Nature 282:39-43 (1979); Kingsman, A. J. , et al . , Gene 7:141-152 (1979); Tschumper, G., et al., Gene 10:157-166

(1980)] . This plasmid already contains the trp gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan [e.g., ATCC 44076 or PEP4-1; Jones, E. W. , Genetics 85:23-33 (1977)]. Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase, which is found on plasmid pAP12BD ATCC 53231 [Patel, A. C, et al . , U.S. Patent No. 4,935,350, issued June 19, 1990] or other glycolytic enzymes such as enolase, which is found on plasmid pACl (ATCC 39532) , glyceraldehyde-3-phosphate dehydrogenase, which is derived from plasmid pHcGAPCl (ATCC 57090, 57091), zymomonas mobilis [Ingram, L.O., et al . , U.S. Patent No. 5,000,000 issued March 19, 1991], hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, which is contained on plasmid vector pCL28XhoLHBPV [ATCC 39475; Reddy, V. B., et al . , U.S. Patent No. 4,840,896, issued June 20, 1989], glyceraldehyde 3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization, such as, the GAL1 promoter, which may be found on plasmid pRY121 (ATCC 37658) . Suitable vectors and promoters for use in yeast expression are further described in Hitzeman, R. A., et al., European Patent Publication No. 73,657A1, published March 9, 1983. Yeast enhancers such as the UAS Gal from Saccharomyces cerevisiae, which is found in conjunction with the CYC1 promoter on plasmid YEpsec--hIlbeta (ATCC 67024) , also are advantageously used with yeast promoters. The following examples are presented to further illustrate the preparation of the claimed proteins. The scope of the present invention is not to be construed as merely consisting of the following examples.

Example 1

Vector Construction

A gene of SEQ ID NO: 14 is assembled from a -220 base pair and a ~240 base pair segment which are derived from chemically synthesized oligonucleotides: 1 CATATGAGGG TACCTATCCA AAAAGTACAA GATGACACCA AAACACTGAT

51 AAAGACAATA GTCACAAGGA TAAATGATAT CTCACACACA CAGTCAGTCT

101 CATCTAAACA GAAAGTCACA GGCTTGGACT TCATACCTGG GCTGCACCCC

151 ATACTGACAT TGTCTAAAAT GGACCAGACA CTGGCAGTCT ATCAACAGAT

201 CTTAACAAGT ATGCCTTCTA GAAACGTGAT ACAAATATCT AACGACCTGG 251 AGAACCTGCG GGATCTGCTG CACGTGCTGG CCTTCTCTAA AAGTTGCCAC 301 TTGCCATGGG CCAGTGGCCT GGAGACATTG GACAGTCTGG GGGGAGTCCT 351 GGAAGCCTCA GGCTATTCTA CAGAGGTGGT GGCCCTGAGC AGGCTGCAGG 401 GGTCTCTGCA AGACATGCTG TGGCAGCTGG ACCTGAGCCC CGGGTGCTAA 451 TAGGATCC (SEQ ID NO: 14) The 220 base pair segment extends from the Ndel site to the Xbal site at position 220 within the coding region and is assembled from 7 overlapping oligonucleotides which range in length from between 34 and 83 bases. The 240 base pair segment which extends from the Xbal to the BamHI site is also assembled from 7 overlapping oligonucleotides which range in length from between 57 and 92 bases. To assemble these fragments, the respective 7 oligonucleotides are mixed in equimolar amounts, usually at concentrations of about 1-2 picomoles per microliter. Prior to assembly, all but the oligonucleotides at the 5' -ends of the segment are phosphorylated in standard kinase buffer with T4 DNA kinase using the conditions specified by the supplier of the reagents. The mixtures are heated to 95°C and allowed to cool slowly to room temperature over a period of 1-2 hours to ensure proper annealing of the oligonucleotides. The oligonucleotides are then ligated to each other and into an appropriated cloning vector such as pUC18 or pUC 19 using T4 DNA ligase. The buffers and conditions are those recommended by the supplier of the enzyme. The vector for the 220 base pair fragment is digested with Ndel and Xbal, whereas the vector for the 240 base pair fragment is digested with Xbal and BamHI prior to use. The ligation mixes are used to transform E. coli DH10B cells (commercially available from Life Technologies, producer of GIBCO/BRL products, Grand Island, NY) and the transformed cells are plated on tryptone-yeast plates (TY,

Difco, Detroit, MI) containing 100 μg/ml of ampicillin, X- gal and IPTG. Colonies which grow up overnight are grown in liquid TY medium with 100 μg/ml of ampicillin and are used for plasmid isolation and DNA sequence analysis . Plasmids with the correct sequence are kept for the assembly of the complete gene. This is accomplished by gel-purification of the 220 base-pair and the 240 base-pair fragments and ligation of these two fragments into an expression vector such as pRB182 from which the coding sequence for A-C-B proinsulin is deleted and is digested with Ndel and BamHI prior to use.

Alternatively, plasmid pET30 (Novagen, Madison, WI) can be digested with Ndel and BamHI, and the desired DNA sequence encoding the proteins of the present invention can be inserted by procedures recognized in the art and described herein. The source of the DNA is synthetic oligonucleotides that are assembled by art recognized methodology and described herein.

Example 2 Expression plasmid for the Protein of SEQ ID NO: 3 (pHS767)

Plasmid vector pOJ722 (40 μg, NRRL No. B-21,654) was prepared in a manner analogous to Example 1 and was digested with 20 units of EcoRV (Boehringer Mannheim, Indianapolis, IN) and 20 units of Bglll (Boehringer Mannheim) in "Boehringer Mannheim buffer B" at 37°C for two hours. The digest was purified on a 1% agarose gel, and the 2674 bp and 1422 bp fragments were isolated using the freeze-squeeze method.

The oligonucleotides 14255 [5'-ATC TCA CAC ACA CAG TCA GTC TCG AGT AAA CAG AAA GTC ACA GGC TTG GAC TTC ATA CCT GGG CTG CAC CCC GAC CTG ACA TTG TCT AAA ATG GAC CAG ACA CTG GCA GTC TAT CAA CA-3' (SEQ ID NO: 15)] and 14256 [5' -GAT CTG TTG ATA GAC TGC CAG TGT CTG GTC CAT TTT AGA CAA TGT CAG GTC GGG GTG CAG CCC AGG TAT GAA GTC CAA GCC TGT GAC TTT CTG TTT ACT CGA GAC TGA CTG TGT GTG TGA GAT-3' (SEQ ID NO: 16)] were kinased in the presence of lx kinase buffer, 50 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 0.5 mM ATP, 1 mM DTT, and 5 units T4 polynucleotide kinase (GIBCO BRL) at 37°C for 30 minutes. The EcoRV/Bglll 2614 bp and 1422 bp fragments, prepared as described above, and the 14255 and 14256 linkers were ligated in the presence of lx kinase buffer, 0.5 mM ATP and 1 unit T4 DNA ligase (GIBCO BRL) at 16°C overnight. The ligation products are transformed into E. coli BL21(DE3) (Novagen) and colonies growing on TY plates supplemented with 10 μg/mL of tetracycline are analyzed. Plasmid DNA is isolated, then subjected to DNA sequencing on a PE-Applied Biosystems 370 DNA sequencer. Plasmids containing the expected -400 bp Ndel to BamHI fragment are kept, and are designated pHS767.

Example 3 Expression plasmid for the Protein of SEQ ID NO: 5 (pHS786)

Plasmid vector pHS767 (40 μg) was digested with 20 units of Pmll (New England Biolab, Beverly, MA) in "New England Biolab buffer 1" at 37°C for two hours. Buffer salts were added to adjust to the mixture to the conditions of "New England Biolab buffer 3." BstXI was added (20 units, New England Biolab) , and the plasmid was digested for 2 hours at 55°C. After that, the digest was treated with 20 units of alkaline phosphatase (Boehringer Mannheim) at 37°C for 30 minutes. The digest was purified on a 1% agarose gel, and the 4170 bp fragment was isolated using the freeze- squeeze method.

The oligonucleotides 13824 [5' -TGA GGC TTC CAG GAC TCC CCC CAG ACT GTC CAA TGT CTC CAG GCC ACT GGC GTC TGG CAA GTG GCA ACT TTT AGA GAA GGC CAG CAC-3' (SEQ ID NO: 17)] and 13825 [5' -GTG CTG GCC TTC TCT AAA AGT TGC CAC TTG CCA GAC GCC AGT GGC CTG GAG ACA TTG GAC AGT CTG GGG GGA GTC CTG GAA GCC-3' (SEQ ID NO:18)] were kinased in the presence of lx kinase buffer, 50 mM Tris-HCl pH 8.0, 10 mM MgCl2, 0.5 mM ATP, 1 mM DTT, and 5 units T4 polynucleotide kinase (GIBCO

BRL) at 37°C for 30 minutes.

The Pmll/BstXI linearized pHS767 vector, and the 13824 and 13825 linkers, were ligated in the presence of lx kinase buffer, 0.5 mM ATP and 1 unit T4 DNA ligase (GIBCO BRL) at 16°C overnight. The ligation products are transformed into E. coli BL21(DE3) (Novagen) and colonies growing on TY plates supplemented with 10 μg/mL of tetracycline are analyzed. Plasmid DNA is isolated, then subjected to DNA sequencing on a PE-Applied Biosystems 370 DNA sequencer. Plasmids containing the expected -400 bp

Ndel to BamHI fragment are kept, and are designated pHS786. Example 4 Expression plasmid for the Protein of SEQ ID NO: 2 (pHS775)

Plasmid vector pHS767 (40 μg) was digested with 20 units of Xhol (Boehringer Mannheim) and 20 units of Ncol (Boehringer Mannheim) in "Boehringer Mannheim buffer H" at 37°C for two hours. The digest was treated with 20 units of alkaline phosphatase (Boehringer Mannheim) at 37°C for 30 minutes. The digest was purified on a 1% agarose gel, and the 4215 bp fragment was isolated using the freeze-squeeze method.

The oligonucleotides 14405 [5'-TCG AGT AAA CAG AAG GTC ACC GGC TTG GAC TTC ATA CCT GGG CTG GAC CCC ATA CTG ACA TTG TCT AAA ATG GAC CAG ACA CTG GCA GTC TAT CAA CA-3' (SEQ ID NO: 19)] and 14406 [5' -GAT CTG TTG ATA GAC TGC CAG TGT CTG GTC CAT TTT AGA CAA TGT CAG TAT GGG GTC CAG CCC AGG TAT GAA GTC CAA GCC GGT GAC CTT CTG TTT AC-3' (SEQ ID NO: 20)] were kinased in the presence of lx kinase buffer, 50 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 0.5 mM ATP, 1 mM DTT, and 5 units T4 polynucleotide kinase (GIBCO BRL) at 37°C for 30 minutes. The Xhol/Ncol linearized pHS767 vector and 14405 and 13406 linkers were ligated in the presence of lx Kinase buffer, 0.5 mM ATP and 1 unit T4 DNA ligase (GIBCO BRL) at 16°C overnight. The ligation products are transformed into E. coli BL21(DE3) (Novagen) and colonies growing on TY plates supplemented with 10 μg/mL of tetracycline are analyzed. Plasmid DNA is isolated, then subjected to DNA sequencing on a PE-Applied Biosystems 370 DNA sequencer. Plasmids containing the expected -400 bp Ndel to BamHI fragment are kept, and are designated pHS775. Example 5

Expression plasmid for the Protein of SEQ ID NO: 4 (pHS785)

Plasmid vector pHS775 (40 μg) was digested with 20 units of Pmll (New England Biolab) in "New England Biolab buffer 1" at 37°C for two hours. The buffer salts were adjusted to "New England Biolab buffer 3," 20 units of BstXI (New England Biolab) were added, and the plasmid was digested for 2 hours at 55°C. After that, the digest was treated with 20 units of alkaline phosphatase (Boehringer Mannheim) at 37°C for 30 minutes. The digest was purified on a 1% agarose gel, and the 4170 bp fragment was isolated using the freeze-squeeze method.

The oligonucleotides 13824 [5' -TGA GGC TTC CAG GAC TCC CCC CAG ACT GTC CAA TGT CTC CAG GCC ACT GGC GTC TGG CAA GTG GCA ACT TTT AGA GAA GGC CAG CAC-3' (SEQ ID NO:21)] and 13825 [5' -GTG CTG GCC TTC TCT AAA AGT TGC CAC TTG CCA GAC

GCC AGT GGC CTG GAG ACA TTG GAC AGT CTG GGG GGA GTC CTG GAA GCC-3' (SEQ ID NO:22)] were kinased in the presence of lx kinase buffer, 50 mM Tris-HCl pH 8.0, 10 mM MgC12, 0.5 mM ATP, 10 mM DTT, and 5 units T4 polynucleotide kinase (GIBCO BRL) .

The Pmll/BstXI linearized pHS775 vector, and the 13824 and 13825 linkers, were ligated in the presence of lx kinase buffer, 0.5 mM ATP and 1 unit T4 DNA ligase (GIBCO BRL) at 16°C overnight. The ligation products are transformed into E. coli BL21(DE3) (Novagen) and colonies growing on TY plates supplemented with 10 μg/mL of tetracycline are analyzed. Plasmid DNA is isolated, then subjected to DNA sequencing on a PE-Applied Biosystems 370 DNA sequencer. Plasmids containing the expected -400 bp Ndel to BamHI fragment are kept, and are designated pHS785.

Example 6 Transformation of E. coli with expression plasmids

The expression plasmids were transformed into E. coli cells. Competent E. coli cells (100 μL) were transformed with 10 ng of the expression plasmids described earlier. The transformation mixtures were plated in TY-agar plates containing tetracycline (10 μg/mL) . Transformants were picked and used to grow 10 L cultures. Cells harboring the expression plasmid for the desired protein were grown overnight at 37°C in 1 L of 2xTY medium containing tetracycline (10 μg/mL) . After overnight growth, 100 mL of a culture were inoculated into 10 L of medium. When the culture optical density reached 0.6 O.D., expression of the particular protein was induced by adding IPTG to a concentration of 10 μM. After 3 hours, the E. coli cells were harvested, and granules were isolated for protein purification.

The techniques of transforming cells with the aforementioned vectors are well-known in the art [Maniatis, T., et al. Molecular Cloning: A Laboratory Manual , 2^nd Ed., Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1988); Id. , Current Protocols in Molecular Biology (1989) , and supplements thereof] . The techniques involved in the transformation of E. coli cells used in the preferred practice of the invention as exemplified herein are well known in the art. The precise conditions under which the transformed E. coli cells are cultured is dependent on the nature of the E. coli host cell line and the expression or cloning vectors employed. For example, vectors which incorporate thermoinducible promoter- operator regions, such as the cl857 thermoinducible lambda- phage promoter-operator region, require a temperature shift from about 30 C to about 40 C. in the culture conditions so as to induce protein synthesis. In the preferred embodiment of the invention, E. coli K12 RV308 cells are employed as host cells, but numerous other cell lines are available, such as, but not limited to, E. coli K12 L201, L687, L693, L507, L640, L641, L695, L814 (E. coli B) . The transformed host cells are then plated on appropriate media under the selective pressure of the antibiotic corresponding to the resistance gene present on the expression plasmid. The cultures are then incubated for a time and temperature appropriate to the host cell line employed. Proteins that are expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies which contain high levels of the overexpressed protein [Kreuger, J. K., et al . , in Protein Folding, Gierasch, L. M. and King, J., eds., American Association for the Advancement of Science Publication No. 89-18S, Washington, D.C., 136-142 (1990)]. Such protein aggregates must be dissolved to provide further purification and isolation of the desired protein product. [Kreuger, J. K. , et al., supra. ] . A variety of techniques using strongly denaturing solutions such as guanidinium-HCl and/or weakly denaturing solutions such as urea are used to solubilize the proteins. Removal of the denaturing agents allows the denatured protein to assume its native conformation. The particular conditions for denaturation and folding are determined by the particular protein expression system and/or the protein in question.

Preferably, the present proteins are expressed with a leader sequence . One of ordinary skill in the art would recognize that numerous leader sequences are operable. However, the leader sequence is preferably Met-Rχ-, wherein Ri is any amino acid except Pro or is absent, so that the expressed proteins may be readily converted to the claimed protein with Cathepsin C, or other suitable aminopeptidases . Preferably, Ri is Arg, Asp, or Tyr; and most preferably, the proteins are expressed with a Met-Arg leader sequence. Interestingly, the leader sequence does not significantly affect stability or activity of the protein. Nevertheless, the leader sequence is preferably cleaved from the protein. Thus, the proteins of the Formula: Met-Rχ-SEQ ID NO:l are useful as biological agents and, preferably, as intermediates . Example 7 The Protein of the Formula: Met-Arg-SEQ ID NO: 4

The protein of SEQ ID NO: 4 with a Met-Arg leader sequence was expressed in E. coli as previously described. Granules were solubilized in 8 M urea containing 5 mM cysteine. The protein was purified by anion exchange in 8 M urea containing 5 mM cysteine and folded by dilution into 8 M urea (containing 5 mM cysteine) and exhaustive dialysis against phosphate-buffered saline (PBS) . Little to no aggregation of the protein was seen. Following final purification of the protein by size exclusion chromatography, the protein was concentrated to 3-3.5 mg/mL in PBS.

Example 8 The Protein of the Formula SEQ ID NO: 3

The protein of SEQ ID NO: 3 with a Met-Arg leader sequence was expressed in E. coli , isolated and folded by techniques analogous to the previous Examples. The pH of the protein solution was reduced to pH 2.8. The Met-Arg leader sequence was cleaved by the addition of 6-10 milliunits dDAP per mg of protein (dDAP is the abbreviation for a dipeptidylaminopeptidase isolated from the slime mold, Dicteostelium descoidium, described by Atkinson, P. R., et al . , U.S. Patent No. 5,565,330, issued October 15, 1996). The conversion reaction was allowed to proceed for 2-8 hours at room temperature. The progress of the reaction was monitored by high performance reversed phase chromatography. The reaction was terminated by adjusting the pH to 8 with NaOH. The des (Met-Arg) protein was further purified by cation exchange chromatography in the presence of 7-8 M urea and size exclusion chromatography in PBS. Following final purification of the proteins by size exclusion chromatography the proteins were concentrated to 3-3.5 mg/mL in PBS . Virtually no aggregation of either protein was noted. The purification of the claimed proteins is by techniques known in the art and includes reversed phase chromatography, affinity chromatography, ion exchange and size exclusion chromatography. The claimed proteins contain two cysteine residues. Thus, a disulfide bond may be formed to stabilize the protein. The present invention includes proteins of the Formula (I) wherein the Cys at position 96 is cross-linked to Cys at position 146 as well as those proteins without such disulfide bonds. Preferably, the Cys at position 96 is disulfide bonded to the Cys at position 146. In addition the proteins of the present invention may exist, particularly when formulated, as dimers, trimers, tetramers, and other multimers . Such multimers are included within the scope of the present invention.

The present invention provides a method for treating obesity. The method comprises administering to the organism an effective amount of anti-obesity protein in a dose between about 1 and 1000 μg/kg. A preferred dose is from about 10 to 100 μg/kg of active protein. A typical daily dose for an adult human is from about 0.5 to 100 mg. In practicing this method, proteins of the Formula (I) can be administered in a single daily dose or in multiple doses per day. The treatment regime may require administration over extended periods of time. The amount per administered dose or the total amount administered will be determined by the physician and depend on such factors as the nature and severity of the disease, the age and general health of the patient and the tolerance of the patient for the protein. The instant invention further provides pharmaceutical formulations comprising proteins of the present invention. The proteins, preferably in the form of a pharmaceutically acceptable salt, can be formulated for parenteral administration for the therapeutic or prophylactic treatment of obesity. For example, proteins of the Formula (I) can be admixed with conventional pharmaceutical carriers and excipients. The compositions comprising claimed proteins contain from about 0.1 to 95% by weight of the active protein, preferably in a soluble form, and more generally from about 10 to 30%. Furthermore, the present proteins may be administered alone or in combination with other anti-obesity agents or agents useful in treating diabetes. For intravenous (i.v.) use, the protein is administered in commonly-used intravenous fluid (s) and administered by infusion. Such fluids, for example, physiological saline, Ringer's solution or 5% dextrose solution can be used. For intramuscular preparations, a sterile formulation, preferably a suitable soluble salt form of a protein of the Formula (I) , for example the hydrochloride salt, can be dissolved and administered in a pharmaceutical diluent such as pyrogen-free, distilled water, physiological saline, or 5% glucose solution. A suitable insoluble form of the protein may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g., an ester of a long chain fatty acid such as ethyl oleate.

Pharmaceutically acceptable preservatives such as an alkylparaben, particularly methylparaben, ethylparaben, propylparaben, or butylparaben or chlorobutanol are preferably added to the formulation to allow multi-dose use. Significantly, the claimed proteins having Asp or Glu at position 100 are also stable in the presence of a phenolic preservative, such as, m-cresol or phenol. Proteins with one of these replacements are highly preferred for that reason. The stability of the proteins in the presence of a phenolic preservative offers advantages in pharmaceutical delivery, including, enhanced preservative effectiveness. The formulation is preferably prepared in the absence of salt to minimize the ionic strength of the formulation. The ability of the present proteins to treat obesity is demonstrated in vivo, as follows. Biological Testing

Parabiotic experiments suggest that a protein is released by peripheral adipose tissue and that the protein is able to control body weight gain in normal, as well as obese mice. Therefore, the most closely related biological test is to inject the test protein by any of several routes of administration (e.g., i.v., s.c, i.p., or by minipump or cannula) and then to monitor food and water consumption, body weight gain, and also plasma chemicals or hormones, such as glucose, insulin, ACTH, corticosterone, GH, or T4 over various time periods . Suitable test animals include normal mice (ICR, etc.) and obese mice (ob/ob, Avy/a, KK-Ay, tubby, fat) . The ob/ob mouse model of obesity and diabetes is generally accepted in the art as being indicative of the obesity condition. Control for non-specific effects for these injections is accomplished by administering the vehicle without the protein in the same animal model, and monitoring the same parameters. Alternatively, the protein itself is administered to animals that are thought to lack the protein receptor, such as, db/db mice or fa/ fa or cp/cp rats. Proteins demonstrating activity in these models will demonstrate similar activity in other mammals, particularly humans .

Since the target tissue is expected to be the hypothalamus where food intake and lipogenic state are regulated, an alternate study involves injecting a protein directly into the brain of test animals, for example, by i.c.v. injection via the lateral or third ventricles, or by injection directly into specific hypothalamic nuclei, such as, the arcuate, paraventricular, or perifornical nuclei.

The same parameters as above could be measured, or the release of neurotransmitters that are known to regulate feeding or metabolism could be monitored (e.g., NPY, galanin, norepinephrine, dopamine, β-endorphin release) . Similar studies are accomplished in vitro using isolated hypothalamic tissue in a perifusion or tissue bath system. In this situation, the release of neurotransmitters or electrophysiological changes is monitored.

The proteins used to carry out the experiments that are reported in Examples 9 and 10 were prepared in accordance with the teachings and examples provided herein. Amino acid sequences of the proteins were confirmed by mass spectroscopy and/or amino acid analysis . The proteins were folded with the Cys residues cross-linked by a disulfide bond when tested. Example 9

Testing of Proteins of SEQ ID NO: 2 and 3 in Male ob/ob Mice

Male ob/ob mice [Harlan, Ltd., Blackthorn, England] were housed in groups of 5 animals each and provided with Purina 5008 chow and water ad libitum. The mice were maintained on a reverse lighting schedule (lights off at 9:00 A.M., and on at 9:00 P.M.). The mice were weighed daily at 8:30 A.M. Their food and water consumption were determined at the same time. Treatment, as indicated below, were made following the morning weighing, just prior to lights out. The mice were treated once daily for 4 days.

The effects of these treatments on food consumption and cumulative body weight change are illustrated for representative proteins of the present invention in Tables 1 and 2. Table 1. Effect of SEQ ID.NO:2 on food consumption and cumulative body weight change in ob/ob mice

Table 2. Effect of SEQ ID NO: 3 on food consumption and cumulative body weight change in ob/ob mice

Group 1 = Control (PBS) ; Group 2 = 30 μg/day of protein; Group 3 = 300 μg/day of protein

Example 10 Analysis of Aggregation by Dynamic Light Scattering (DLS)

The physical properties of the present proteins are demonstrated as follows . Based on availability of material, the solution analyzed by DLS was prepared in one of two ways. Material from the last size exclusion chromatography purification step, at approximately 1.5 mg/mL in PBS, was either concentrated to about 3 or 5 mg/mL by diafiltration, or was dialyzed against water, lyophilized, and then reconstituted to about 3 or 5 mg/mL.

In the first method the protein solution at approximately 1.5 mg/mL was concentrated to greater than about 3 or 5 mg/mL in a small volume stir cell (10 mL) using an Amicon YM10, 25-mm membrane. This was conducted under cold room conditions (about 5°C) . Protein concentration was determined by UV absorption, and the solution was diluted to about 3.0 mg/mL or 5 mg/mL. with PBS (10-fold water dilution of lOx PBS without Ca/Mg, GIBCO BRL) .

A second alternative method, used when lyophilization was feasible, consisted of dialyzing the solution of protein against water, using three to five exchanges of water at about 4 C. A typical dialysis membrane was the Spectra/Por-7 dialysis membrane, 2000 molecular weight cutoff membrane (Spectrum Medical Industries, Los Angeles, CA) . The material was concentrated as above to 3 to 4 mg/mL and typically a 2 mg quantity was lyophilized in a 5 mL vial. For DLS analysis this plug was reconstituted with water to greater than 3.3 mg/mL or greater than 5 mg/mL. Several vials were typically pooled. Protein concentration was determined by UV at peak maximum (typically 280-nm) . The protein concentration was diluted to about 3 or 5 mg/mL with a combination of water and lOx PBS (without Ca/Mg, GIBCO BRL) to yield a final PBS concentration of lx.

The protein solution at 3.0 mg/mL or at 5 mg/mL in lx PBS was adjusted to 7.4 with HCl/NaOH and passed through a 0.1 μm Anotop™-10 filter (Whatman International, Ltd., Maidstone, England) into a DLS cell. The average cumulant particle size was measured on a Brookhaven BI-9000 DLS instrument (Brookhaven Instruments, Holtsville, NY) with a Lexel argon-ion laser every 15 minutes using a 30 second duration at a 90° angle. A 488-nm filter with a 400 μm pinhole was assumed. At the 37°C incubation temperature a viscosity of 0.6915 centipoise (cP) and refractive index of 1.333 was used. The light-weighted average particle size was calculated by a cumulant method using the measured autocorrelation baseline.

The estimated time required for various anti- obesity proteins to reach an averaged light-weighted particle size of 50 nm in a PBS solution at pH 7.4 and 37°C is shown in Table 3. The averaged light-weighted particle size was determined from a cumulant analysis of a binodal distribution composed of monomeric and higher- order aggregate populations. The time necessary to achieve an average aggregate size of 50 nm was estimated by plotting size as a function of time. The proteins were folded with the Cys residues cross-linked by a disulfide bond when tested.

Table 3. Estimated time (minutes) required for various anti-obesity proteins to reach an averaged light-weighted particle size of 50 nm in a PBS solution at pH 7.4 and 37°C.

The data in Table 3 demonstrate the dramatic reduction in the propensity of the claimed proteins to aggregate .

The claimed proteins are active in at least one of the above biological tests and are anti-obesity agents. As such, they are useful in treating obesity and those disorders implicated by obesity. However, the proteins are not only useful as therapeutic agents. One skilled in the art recognizes that the proteins are useful in the production of antibodies for diagnostic use and, as proteins, are useful as feed additives for animals. Furthermore, the proteins are useful for controlling weight for cosmetic purposes in mammals. A cosmetic purpose seeks to control the weight of a mammal to improve bodily appearance. The mammal is not necessarily obese. Such cosmetic use forms part of the present invention.

The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected is not limited to the particular forms disclosed, since they are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art without departing from the spirit of the invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Beals, John M

Hoffmann, James A Kline, Allen D Schevitz, Richard W Zhang, Faming

(ii) TITLE OF INVENTION: Anti-Obesity Proteins

(iii) NUMBER OF SEQUENCES: 22

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Eli Lilly and Company

(B) STREET: Lilly Corporate Center

(C) CITY: Indianapolis (D) STATE: Indiana

(E) COUNTRY: U.S.A.

(F) ZIP: 46285

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: WO N/A (W/ Filing)

(B) FILING DATE:

(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Caltrider, Steven P

(B) REGISTRATION NUMBER: 36,467

(C) REFERENCE/DOCKET NUMBER: X-11092 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (317) 276-0757

(B) TELEFAX: (317) 277-1917

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 146 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Xaa at position 1 is Val or absent;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2 (D) OTHER INFORMATION: /note= "Xaa at position 2 is Pro or absent ; "

( ix) FEATURE :

(A) NAME/KEY: Modified-site (B) LOCATION: 22

(D) OTHER INFORMATION: /note= "Xaa at position 22 is Asn or Ser; "

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 28

(D) OTHER INFORMATION: /note= "Xaa at position 28 is Gin or absent ; " (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 72

(D) OTHER INFORMATION: /note= "Xaa at position 72 is Asn, Gin, Glu or Asp; "

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 73

(D) OTHER INFORMATION: /note= "Xaa at position 73 is Val or Met;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 100 (D) OTHER INFORMATION: /note= "Xaa at position 100 is Trp,

Gin, Glu, Asp, Ser, Thr, Lys, His, or ..."

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 138

(D) OTHER INFORMATION: /note= "Xaa at position 138 is Trp, Gin, Glu, Asp, Ser, Thr, Lys, His, or Arg; said protein having at least one of the following:" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 1

(D) OTHER INFORMATION: /note= "Xaa at position 1 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 2

(D) OTHER INFORMATION: /note= "Xaa at position 2 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 3 (D) OTHER INFORMATION: /note= "He at position 3 is replaced with Glu, Asp, Arg, Lys, or His;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 30

(D) OTHER INFORMATION: /note= "Val at position 30 is replaced with Glu, Asp, Arg, Lys, or His;"

(ix) FEATURE: (A) NAME/KEY: Modified-site (B) LOCATION: 36

(D) OTHER INFORMATION : /note= "Val at position 36 is replaced with Glu, Asp, Arg, Lys , or His ; "

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 41

(D) OTHER INFORMATION: /note= "Phe at position 41 is replaced with Glu, Asp, Arg, Lys, or His;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 42

(D) OTHER INFORMATION: /note= "He at position 42 is replaced with Glu, Asp, Arg, Lys, or His;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 43 (D) OTHER INFORMATION: /note= "Pro at position 43 is replaced with Glu, Asp, Arg, Lys, or His;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 45

(D) OTHER INFORMATION: /note= "Leu at position 45 is replaced with Glu, Asp, Arg, Lys, or His;"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 46

(D) OTHER INFORMATION: /note= "His at position 46 is replaced with Glu, Asp, Arg, or Lys;" (ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 47

(D) OTHER INFORMATION: /note= "Pro at position 47 is replaced with Glu, Asp, Arg, Lys, or His;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 48

(D) OTHER INFORMATION: /note= "He at position 48 is replaced with Glu, Asp, Arg, Lys, or His;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 49 (D) OTHER INFORMATION: /note= "Leu at position 49 is replaced with Glu, Asp, Arg, Lys, or His;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 50

(D) OTHER INFORMATION: /note= "Thr at position 50 is replaced with Glu, Asp, Arg, Lys, or His;"

(ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 74

(D) OTHER INFORMATION: /note= "He at position 74 is replaced with Gin, Gly, Asp, Arg, Lys, His, Thr or Ser;" (ix) FEATURE: (A) NAME/KEY: Modified-site

(B) LOCATION: 89

(D) OTHER INFORMATION: /note= "Val at position 89 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 92

(D) OTHER INFORMATION: /note= "Phe at position 92 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser;"

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 99 (D) OTHER INFORMATION: /note= "Pro at position 99 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; or"

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 142

(D) OTHER INFORMATION: /note= "Leu at position 142 is replaced with Glu, Asp, Arg, Lys, or His; or a pharmaceutically acceptable salt thereof . "

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Xaa Xaa He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15

He Val Thr Arg He Xaa Asp He Ser His Thr Xaa Ser Val Ser Ser 20 25 30

Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 35 40 45

Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 50 55 60 Leu Thr Ser Met Pro Ser Arg Xaa Xaa He Gin He Ser Asn Asp Leu 65 70 75 80

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 85 90 95

His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110

Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125

Leu Gin Gly Ser Leu Gin Asp Met Leu Xaa Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 146 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15

He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser 20 25 30

Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu Asp Pro He 35 40 45

Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 50 55 60

Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 65 70 75 80

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 85 90 95

His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly

100 105 110

Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125

Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145

(2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 146 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15

He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser 20 25 30

Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro Asp 35 40 45

Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 50 55 60 Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 65 70 75 80

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 85 90 95

His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110

Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125

(2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 146 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15

He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser 20 25 30

Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu Asp Pro He 35 40 45

Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 50 55 60

Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 65 70 75 80

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 85 90 95

His Leu Pro Asp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly

100 105 110

Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125

(2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15

He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser 20 25 30

Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro Asp 35 40 45

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 85 90 95

His Leu Pro Asp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110

Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 146 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15

He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser 20 25 30

Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 35 40 45 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He 50 55 60

Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn Asp Leu 65 70 75 80

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Asp Ser Lys Ser Cys 85 90 95

His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110

Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125

(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 146 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr 1 5 10 15

He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val Ser Ser 20 25 30

Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He 35 40 45

Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Asp Ser Lys Ser Cys 85 90 95

His Leu Pro Asp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110

Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125

Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 148 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Met Arg Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He 1 5 10 15

Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val 20 25 30

Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu Asp 35 40 45

Pro He Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin 50 55 60 Gin He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn 65 70 75 80

Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys 85 90 95

Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu 100 105 110

Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu 115 120 125

Ser Arg Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu 130 135 140 Ser Pro Gly Cys 145

(2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 148 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

( ii ) MOLECULE TYPE : protein

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 9 :

Met Arg Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He 1 5 ιo 15 Lys Thr He Val Thr Arg lie Asn Asp He Ser His Thr Gin Ser Val 20 25 30

Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His 35 40 45

Pro Asp Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin 50 55 60 Gin He Leu Thr Ser Met Pro Ser Arg Asn Val He Gin He Ser Asn 65 70 75 80

Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys 85 90 95

Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu 100 105 110

Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu 115 120 125

(2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 148 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Met Arg Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He 1 5 10 15

Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val 20 25 30

Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu Asp 35 40 45

Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys 85 90 95

Ser Cys His Leu Pro Asp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu 100 105 110

Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu 115 120 125 Ser Arg Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu 130 135 140 Ser Pro Gly Cys 145

(2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 148 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Met Arg Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He 1 5 10 15

Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val 20 25 30

Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His 35 40 45

Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys 85 90 95

Ser Cys His Leu Pro Asp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu 100 105 110

Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu 115 120 125

(2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 148 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Met Arg Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He 1 5 10 15

Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val 20 25 30

Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His 35 40 45

Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Asp Ser Lys 85 90 95

Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu 100 105 110

Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu 115 120 125

(2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 148 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Met Arg Val Pro He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He 1 5 10 15

Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr Gin Ser Val 20 25 30

Ser Ser Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His 35 40 45

Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Asp Ser Lys 85 90 95 Ser Cys His Leu Pro Asp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu 100 105 110

Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu 115 120 125

(2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 458 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

CATATGAGGG TACCTATCCA AAAAGTACAA GATGACACCA AAACACTGAT AAAGACAATA 60

GTCACAAGGA TAAATGATAT CTCACACACA CAGTCAGTCT CATCTAAACA GAAAGTCACA 120

GGCTTGGACT TCATACCTGG GCTGCACCCC ATACTGACAT TGTCTAAAAT GGACCAGACA 180

CTGGCAGTCT ATCAACAGAT CTTAACAAGT ATGCCTTCTA GAAACGTGAT ACAAATATCT 240 AACGACCTGG AGAACCTGCG GGATCTGCTG CACGTGCTGG CCTTCTCTAA AAGTTGCCAC 300

TTGCCATGGG CCAGTGGCCT GGAGACATTG GACAGTCTGG GGGGAGTCCT GGAAGCCTCA 360

GGCTATTCTA CAGAGGTGGT GGCCCTGAGC AGGCTGCAGG GGTCTCTGCA AGACATGCTG 420

TGGCAGCTGG ACCTGAGCCC CGGGTGCTAA TAGGATCC 458

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 119 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: ATCTCACACA CACAGTCAGT CTCGAGTAAA CAGAAAGTCA CAGGCTTGGA CTTCATACCT 60

GGGCTGCACC CCGACCTGAC ATTGTCTAAA ATGGACCAGA CACTGGCAGT CTATCAACA 119

(2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 123 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

GATCTGTTGA TAGACTGCCA GTGTCTGGTC CATTTTAGAC AATGTCAGGT CGGGGTGCAG 60

CCCAGGTATG AAGTCCAAGC CTGTGACTTT CTGTTTACTC GAGACTGACT GTGTGTGTGA 120

GAT 123

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 87 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

TGAGGCTTCC AGGACTCCCC CCAGACTGTC CAATGTCTCC AGGCCACTGG CGTCTGGCAA 60 GTGGCAACTT TTAGAGAAGG CCAGCAC 87

(2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 84 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

GTGCTGGCCT TCTCTAAAAG TTGCCACTTG CCAGACGCCA GTGGCCTGGA GACATTGGAC 60

AGTCTGGGGG GAGTCCTGGA AGCC 84

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 98 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

TCGAGTAAAC AGAAGGTCAC CGGCTTGGAC TTCATACCTG GGCTGGACCC CATACTGACA 60

TTGTCTAAAA TGGACCAGAC ACTGGCAGTC TATCAACA 98 (2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHHARACTERISTICS :

(A) LENGTH: 98 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: GATCTGTTGA TAGACTGCCA GTGTCTGGTC CATTTTAGAC AATGTCAGTA TGGGGTCCAG 60

CCCAGGTATG AAGTCCAAGC CGGTGACCTT CTGTTTAC 98

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 87 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TGAGGCTTCC AGGACTCCCC CCAGACTGTC CAATGTCTCC AGGCCACTGG CGTCTGGCAA 60

GTGGCAACTT TTAGAGAAGG CCAGCAC 87

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 84 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

GTGCTGGCCT TCTCTAAAAG TTGCCACTTG CCAGACGCCA GTGGCCTGGA GACATTGGAC 60 AGTCTGGGGG GAGTCCTGGA AGCC 84

Claims

We claim:

1. A protein of the Formula (I) :

5 10 15 Xaa Xaa He Gin Lys Val Gin Asp Asp Thr Lys Thr Leu He Lys Thr

20 25 30

He Val Thr Arg He Xaa Asp He Ser His Thr Xaa Ser Val Ser Ser 35 40 45

Lys Gin Lys Val Thr Gly Leu Asp Phe He Pro Gly Leu His Pro He

50 55 60

Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin He

65 70 75 80

Leu Thr Ser Met Pro Ser Arg Xaa Xaa He Gin He Ser Asn Asp Leu

85 90 95 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys

100 105 110

His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125

Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg

130 135 140

Leu Gin Gly Ser Leu Gin Asp Met Leu Xaa Gin Leu Asp Leu Ser Pro

145 Gly Cys (SEQ ID NO:l) (I) wherein:

Xaa at position 1 is Val or absent; Xaa at position 2 is Pro or absent;

Xaa at position 22 is Asn or Ser;

Xaa at position 28 is Gin or absent;

Xaa at position 72 is Asn, Gin, Glu or Asp;

Thr, Lys, His, or Arg;

Xaa at position 1 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg;

Xaa at position 2 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg; lie at position 3 is replaced with Glu, Asp, Arg, Lys, or His;

Val at position 30 is replaced with Glu, Asp, Arg, Lys, or His; Val at position 36 is replaced with Glu, Asp, Arg,

Lys, or His;

Phe at position 41 is replaced with Glu, Asp, Arg, Lys, or His; lie at position 42 is replaced with Glu, Asp, Arg, Lys, or His;

Pro at position 43 is replaced with Glu, Asp, Arg, Lys, or His;

Pro at position 47 is replaced with Glu, Asp, Arg, Lys, or His; lie at position 48 is replaced with Glu, Asp, Arg, Lys, or His;

Leu at position 49 is replaced with Glu, Asp, Arg, Lys, or His;

Thr at position 50 is replaced with Glu, Asp, Arg, Lys, or His; lie at position 74 is replaced with Gin, Glu, Asp,

Arg, Lys, His, Thr or Ser;

Val at position 89 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser;

Phe at position 92 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser;

Pro at position 99 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; or

Leu at position 142 is replaced with Glu, Asp, Arg, Lys, or His; or a pharmaceutically acceptable salt thereof.

2. The protein, of Claim 1 , wherein the Cys at position 96 is disulfide bonded to Cys at position 146.

3. The protein of Claim 2 , wherein Xaa at position 1 is Val;

Xaa at position 2 is Pro;

Xaa at position 28 is Gin or absent;

Xaa at position 72 in Asn or Asp;

Xaa at position 100 is Trp, Glu, or Asp; Xaa at position 138 is Trp, Glu, or Asp; said protein having at least one of the following substitutions :

Phe at position 92 is replaced with Glu, Asp, Arg, Lys, or His.

4. The protein of Claim 3 , wherein Xaa at position 100 is Trp or Asp, and Xaa at position 138 is Trp.

5. The protein of Claim 2 which has a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

6. A protein consisting of the protein as claimed in Claim 1, and a leader sequence, wherein, the leader sequence is bonded to the N-terminal of the protein, or a pharmaceutically acceptable salt thereof.

7. The protein of Claim 6, wherein the leader sequence is Met-R-^ wherein R is absent or is any amino acid except Pro.

8. The protein of Claim 7 , wherein the leader sequence is Met-Arg, Met-Asp, or Met-Tyr.

9. The protein of Claim 8 which has a sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID

NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.

10. A pharmaceutical formulation, comprising as an active agent, a protein of Claim 1, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable diluents, carriers or excipients therefor.

11. A DNA polynucleotide compound comprising DNA that encodes a protein of Claim 1.

12. A DNA polynucleotide compound comprising DNA that encodes a protein of Claim 6.

13. A process for preparing a protein as claimed in Claim 6, comprising:

(a) transforming a host cell with DNA that encodes the protein; (b) culturing the transformed host cell such that the protein is expressed; and

(c) recovering the protein.

14. A process for preparing a protein of Claim 1, comprising:

(a) transforming a host cell with DNA that encodes a protein comprised of a protein of Claim 1 and a leader sequence ;

(b) culturing the transformed host cell such that the protein encoded for in step (a) is expressed;

(c) enzymatically removing the leader sequence of the expressed protein to produce a protein of Claim 1; and (d) recovering the protein of Claim 1.

15. A method of treating obesity or a condition ; associated with obesity, which comprises administering to a mammal in need thereof a protein of the Formula (I) .